An anti-GPRC5D antibody and its multispecific antibody and related applications

Abstract

The present invention discloses an anti-GPRC5D antibody and a multi-specific antibody and related applications, and relates to the field of biomedicine. The anti-GPRC5D antibody or an antigen-binding fragment thereof provided by the present invention comprises CDRs shown in SEQ ID NOs: 1-3, 5-6, SAS or CDRs shown in SEQ ID NOs: 9-11, 13, 14, GTS, and the antibody or antigen-binding fragment thereof has good reactivity and killing activity against multiple myeloma cell lines, and the multi-specific antibody constructed therefrom has good anti-tumor effect, and can be used to prepare related products for preventing, diagnosing or treating tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-GPRC5D antibody and a multispecific antibody thereof and related applications. Background Art

[0002] Multiple myeloma (MM) is a blood tumor characterized by abnormal proliferation of plasma cells (PCs) and the production of a large amount of pathological immunoglobulins in the body. Its incidence is second only to lymphoma among blood tumors, accounting for about 10% of blood system malignancies, with a male-to-female ratio of 1.6:1, and most patients are >40 years old. In the past two decades, with the introduction of immunomodulatory drugs (lenalidomide, pomalidomide), proteasome inhibitors (bortezomib, carfilzomib, ixazomib) and histone deacetylase inhibitors, the treatment of MM has made significant progress. Since the late 1980s, these advances have almost doubled the 5-year survival rate of MM patients.

[0003] GPRC5D (G protein-coupled receptor class C group 5member D) is a 7-transmembrane protein discovered at the beginning of this century. It is an orphan receptor of the G protein-coupled receptor (GPCR) C5 family. GPRC5D mRNA is highly expressed in multiple myeloma cell lines, while GPRC5D mRNA is not highly expressed in any normal tissue except hair follicles, which makes it a potential effective target in multiple myeloma immunotherapy. Its expression on multiple myeloma cells is not related to BCMA expression, and because it is a 7-transmembrane protein, it is not easy to fall off in the serum, but this phenomenon exists in other surface antigens (such as BCMA, CD38) and may lead to reduced efficacy or drug resistance.

[0004] Although the diagnosis and treatment of MM has made significant progress and gradually caught up with international standards, MM is still a non-curable disease, and most patients will eventually relapse, and its treatment still faces great challenges. Therefore, in order to improve the problem of reduced efficacy during treatment, it is necessary to develop safer, more effective, and more diverse new drugs.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-GPRC5D antibody and a multispecific antibody thereof and related applications.

[0007] The present invention is achieved in that:

[0008] In the first aspect, an embodiment of the present invention provides an anti-GPRC5D antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises: CDR1, CDR2 and CDR3 whose amino acid sequences are sequentially shown in SEQ ID NOs: 1, 2 and 3, or CDR1, CDR2 and CDR3 whose amino acid sequences are sequentially shown in SEQ ID NOs: 9, 10 and 11. The light chain variable region comprises: CDR1, CDR2 and CDR3 whose amino acid sequences are sequentially shown in SEQ ID NOs: 5, SAS and SEQ ID NOs: 6, or CDR1, CDR2 and CDR3 whose amino acid sequences are sequentially shown in SEQ ID NOs: 13, GTS and SEQ ID NOs: 14.

[0009] In a second aspect, an embodiment of the present invention provides a multispecific antibody or an antigen-binding fragment thereof, comprising: a first antigen domain that binds to a first target, the multispecific antibody or the antigen-binding fragment thereof further comprising a second antigen domain that binds to a second target and / or a third antigen domain that binds to a third target; the first target comprises GPRC5D, the first antigen domain comprises a heavy chain variable region a and a light chain variable region a; the heavy chain variable region a is the heavy chain variable region described in the aforementioned embodiments, and the light chain variable region a is the light chain variable region described in the aforementioned embodiments.

[0010] In a third aspect, an embodiment of the present invention provides an isolated nucleic acid, an expression cassette containing the isolated nucleic acid, or a recombinant vector containing the isolated nucleic acid, wherein the isolated nucleic acid encodes the anti-GPRC5D antibody or its antigen-binding fragment as described in the preceding embodiments or the multispecific antibody or its antigen-binding fragment as described in the preceding embodiments.

[0011] In a fourth aspect, an embodiment of the present invention provides a host cell containing the recombinant vector described in the aforementioned embodiment.

[0012] In a fifth aspect, an embodiment of the present invention provides an immunoconjugate or a pharmaceutical composition, comprising: an anti-GPRC5D antibody or an antigen-binding fragment thereof as described in the preceding embodiments or a multispecific antibody or an antigen-binding fragment thereof as described in the preceding embodiments.

[0013] In a sixth aspect, embodiments of the present invention provide the use of the anti-GPRC5D antibody or antigen-binding fragment thereof as described in the preceding embodiments, or the multispecific antibody or antigen-binding fragment thereof as described in the preceding embodiments, or the isolated nucleic acid as described in the preceding embodiments, the expression cassette containing the isolated nucleic acid, or the recombinant vector containing the isolated nucleic acid, or the recombinant cell as described in the preceding embodiments, or the immunoconjugate or pharmaceutical composition as described in the preceding embodiments in the preparation of a product for preventing or treating tumors.

[0014] The present invention has the following beneficial effects:

[0015] The present invention provides an anti-GPRC5D antibody or an antigen-binding fragment thereof, which can specifically bind to GPRC5D and has good reactivity and killing activity against multiple myeloma cell lines. The multi-specific antibody constructed therefrom has good anti-tumor effect and can be used to prepare related products for preventing, diagnosing or treating tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 This is a diagram showing the binding effect of the recombinant antibody and GPRC5D+-Hela cells detected by immunofluorescence (IFA) in an embodiment of the present invention;

[0018] Figure 2 This is a diagram of the binding effect of a single-chain antibody and a multiple myeloma cell line analyzed by flow cytometry (FACS) in an embodiment of the present invention;

[0019] Figure 3 This is a graph showing the killing efficiency of ScFv-OKT3 bispecific antibody (BiTE) on target cells detected in an embodiment of the present invention;

[0020] Figure 4 This is a schematic diagram of the construction of the trispecific antibodies K1H1 and K2H2 in the embodiments of the present invention;

[0021] Figure 5 The figure shows the purification of the trispecific antibodies K1H1 and K2H2 in the embodiment of the present invention; wherein a is a real-time monitoring diagram of the purification process, b is an SDS-PAGE diagram, M is a marker, 1 is non-reduced K1H1, 2 is reduced K1H1, 3 is non-reduced K2H2, and 4 is reduced K2H2;

[0022] Figure 6 This is the binding analysis of the trispecific antibodies to human GPRC5D in the examples of the present invention;

[0023] Figure 7 This is an in vitro killing activity analysis of the trispecific antibodies in the examples of the present invention on GPRC5D+-Hela cells, BCMA+-Hela cells and wild-type Hela cells;

[0024] Figure 8 This is an analysis of the survival of the MM mouse transplant model with the trispecific antibodies K1H1 and K2H2 in the examples of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0026] In one aspect, an embodiment of the present invention provides an anti-GPRC5D antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region;

[0027] The heavy chain variable region comprises: CDR1, CDR2 and CDR3 whose amino acid sequences are sequentially shown in SEQ ID NOs: 1, 2 and 3, or CDR1, CDR2 and CDR3 whose amino acid sequences are sequentially shown in SEQ ID NOs: 9, 10 and 11;

[0028] The light chain variable region includes: CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO:5, SAS and SEQ ID NO:6, or CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO:13, GTS and SEQ ID NO:14.

[0029] The antibodies with the above amino acid sequences have good reactivity with multiple myeloma cell lines and have strong killing activity against natural multiple myeloma cell lines. In particular, antibodies with heavy chain variable region CDR1, heavy chain variable region CDR2 and heavy chain variable region CDR3 as shown in the amino acid sequences of SEQ ID No: 9, SEQ ID No: 10 and SEQ ID No: 11 and light chain variable region CDR1, light chain variable region CDR2 and light chain variable region CDR3 as shown in the amino acid sequences of SEQ ID No: 13, GTS and SEQ ID No: 14 have better killing activity against multiple myeloma cells.

[0030] In some embodiments, the heavy chain variable region and the light chain variable region further comprise a framework region.

[0031] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4.

[0032] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:12.

[0033] In some embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:7.

[0034] In some embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:15.

[0035] In some embodiments, the antibody is selected from any one of a monovalent antibody, a multivalent antibody, a multispecific antibody, a chimeric antibody, and a fusion antibody.

[0036] A "multivalent antibody" herein is a polymer of monovalent antibodies that recognize the same epitope and have a higher antigen affinity than the monovalent antibody.

[0037] The "multi-specific antibody" in this article is a polymer of monovalent antibodies that recognize different epitopes, can bind to different targets or different epitopes of the same target, and has higher antigen recognition ability than monovalent antibodies. Fusion antibodies include but are not limited to new fusion molecules formed by combining with other structures (such as BSA, IgG-Fc, etc.) through genetic engineering technology, such as enzymes, antimicrobial peptides or imaging substances that can extend their half-life.

[0038] The "chimeric antibody" herein is an antibody formed by fusing the variable region of a non-human antibody with the constant region or framework region of a human antibody, which can reduce the immune response induced by the non-human antibody.

[0039] In some embodiments, the multispecific antibody comprises any one of a bispecific antibody, a trispecific antibody and a tetraspecific antibody.

[0040] In some embodiments, the antigen-binding fragment includes any one of Fab, Fab', F(ab')2, scFv, and Fv, as long as they exhibit the desired antigen-binding activity.

[0041] The "antigen-binding fragment" herein is also a functional fragment of an antibody, which generally has the same binding specificity as the antibody from which it is derived. It is easy for a person skilled in the art to understand based on the contents described in the present invention that an antigen-binding fragment of an antibody can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of a complete antibody disclosed in the present invention, a person skilled in the art can easily obtain an antigen-binding fragment thereof.

[0042] The antigen-binding fragments can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0043] In some embodiments, when the antigen-binding fragment is scFv, the amino acid sequence of scFv is as shown in SEQ ID NO: 8 or 16.

[0044] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region.

[0045] On the other hand, an embodiment of the present invention provides a multispecific antibody or an antigen-binding fragment thereof, which includes: a first antigen domain that binds to a first target; the multispecific antibody or the antigen-binding fragment thereof also includes a second antigen domain that binds to a second target and / or a third antigen domain that binds to a third target.

[0046] The first target includes GPRC5D, and the first antigen domain includes a heavy chain variable region a and a light chain variable region a; the heavy chain variable region a is the heavy chain variable region described in any of the foregoing embodiments, and the light chain variable region a is the light chain variable region described in any of the foregoing embodiments.

[0047] In some embodiments, the second target comprises BCMA, and the second antigen domain comprises a heavy chain variable region b that binds BCMA.

[0048] In some embodiments, the amino acid sequence of the heavy chain variable region b is as shown in SEQ ID NO:17.

[0049] In some embodiments, the third target comprises CD3.

[0050] In some embodiments, the third antigenic domain comprises the heavy chain variable region c.

[0051] In some embodiments, the third antigen domain also includes a light chain variable region c.

[0052] In some embodiments, the third antigenic domain comprises a single chain antibody that binds CD3.

[0053] In some embodiments, the amino acid sequence of the single-chain antibody (scFv) binding to CD3 is shown in SEQ ID NO:18.

[0054] In some embodiments, the third antigen domain also includes a light chain variable region c.

[0055] In some embodiments, the number of the first antigenic domain, the second antigenic domain and the third antigenic domain is independently 1, 2 or more.

[0056] In some embodiments, the multispecific antibody or antigen-binding fragment thereof further comprises: a heavy chain constant region and / or a light chain constant region.

[0057] In some embodiments, the heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0058] In some embodiments, the heavy chain constant region is derived from cattle, horses, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese or humans. The heavy chain constant region of human origin has a stronger binding ability to T cells.

[0059] In some embodiments, the heavy chain constant region is a heavy chain constant region of human IgG4 or a mutation thereof. Using the heavy chain constant region of IgG4 as the constant region of a multispecific antibody has a very high inhibition rate on target cells and can maintain good binding activity with antigens. The bispecific antibody in the embodiment of the present invention carries a human Fc fragment, retains the binding effect with FcRn, and thus has a longer half-life.

[0060] In some embodiments, the heavy chain constant region includes: a knob chain and / or a hole chain.

[0061] The knob chain is the heavy chain constant region of hIgG4 with knob mutations including S228P, F234A, L235A and T366W.

[0062] The hole chain is the heavy chain constant region of hIgG4 with hole mutations, including S228P, F234A, L235A, T366S, L368A and Y407V.

[0063] In some embodiments, the amino acid sequence of the knob chain is as shown in SEQ ID NO: 21 (patent application number: 202211168052.0).

[0064] In some embodiments, the amino acid sequence of the hole chain is shown in SEQ ID NO: 22 (patent application number: 202211168052.0).

[0065] In some embodiments, the multispecific antibody is selected from the group consisting of: a bispecific antibody and a trispecific antibody.

[0066] In some embodiments, the multispecific antibody comprises: an a chain and a b chain.

[0067] The a chain includes the first antigenic domain and the heavy chain constant region, and the heavy chain constant region is arranged at the C-terminus of the first antigenic domain. The b chain includes the second antigenic domain, the third antigenic domain and the heavy chain constant region, and the second antigenic domain is directly connected to the third antigenic domain or through a linker (e.g. (G4S) n , n is a non-zero natural number selected from 1 to 20, specifically 3 or 4), and the heavy chain constant region is arranged at the C-terminus of the third antigen domain.

[0068] The heavy chain constant region of the a chain is capable of forming a dimer with the heavy chain constant region of the b chain.

[0069] In some embodiments, the heavy chain constant region of the a chain is a hole chain, and the heavy chain constant region of the b chain is a knob chain; or the heavy chain constant region of the a chain is a knob chain, and the heavy chain constant region of the b chain is a hole chain.

[0070] In some embodiments, the amino acid sequence of the a chain is shown in SEQ ID NO:23, and the amino acid sequence of the b chain is shown in SEQ ID NO:24.

[0071] The a chain shown in SEQ ID No: 23 is the anti-GPRC5D antibody shown in SEQ ID No: 16 + the hole chain shown in SEQ ID No: 22 (patent application number: 202211168052.0), referred to as the h1 chain in this article.

[0072] The b chain shown in SEQ ID No: 24 is the humanized anti-BCMA nanobody shown in SEQ ID No: 17 (patent application number: 202211168052.0) + SEQ ID No: 18 anti-CD3 OKT3 antibody + knob chain shown in SEQ ID No: 21 (patent application number: 202211168052.0), referred to as k1 chain in this article.

[0073] The combination of K1 and H1 chains forms a heterodimeric trispecific antibody (K1H1).

[0074] In some embodiments, the multispecific antibody comprises: a c chain and a d chain; wherein the c chain comprises the first antigenic domain, the second antigenic domain and a heavy chain constant region, the heavy chain constant region is arranged at the C-terminus of the first antigenic domain, and the second antigenic domain is arranged at the C-terminus of the heavy chain constant region. The d chain comprises the first antigenic domain, the third antigenic domain and a heavy chain constant region, the heavy chain constant region is arranged at the C-terminus of the third antigenic domain, the first antigenic domain is directly connected to the third antigenic domain or through a linker (for example (G4S)n, n is a non-zero natural number selected from 1 to 20, specifically 3 or 4); the heavy chain constant region of the c chain can form a dimer with the heavy chain constant region of the d chain.

[0075] In some embodiments, the heavy chain constant region of the c chain is a hole chain, and the heavy chain constant region of the d chain is a knob chain; or the heavy chain constant region of the d chain is a knob chain, and the heavy chain constant region of the c chain is a hole chain.

[0076] In some embodiments, the amino acid sequence of the c chain is shown in SEQ ID NO:25, and the amino acid sequence of the d chain is shown in SEQ ID NO:26.

[0077] The amino acid sequence of the c chain is shown in SEQ ID No: 25, which is composed of the anti-GPRC5D antibody shown in SEQ ID No: 16 + the hole chain shown in SEQ ID No: 22 (patent application number: 202211168052.0) + the anti-BCMA antibody shown in SEQ ID No: 17 (patent application number: 202211168052.0), referred to herein as the h2 chain.

[0078] The amino acid sequence of the d peptide chain is shown in SEQ ID No: 26, which is composed of the anti-GPRC5D antibody shown in SEQ ID No: 16 + OKT3 shown in SEQ ID No: 18 + knob chain shown in SEQ ID No: 21 (patent application number: 202211168052.0), referred to herein as k2 chain.

[0079] The combination of H2 chain and K2 chain forms a heterodimeric trispecific antibody (K2H2).

[0080] On the other hand, an embodiment of the present invention provides an isolated nucleic acid, an expression cassette containing the isolated nucleic acid, or a recombinant vector containing the isolated nucleic acid, wherein the isolated nucleic acid encodes an anti-GPRC5D antibody or an antigen-binding fragment thereof as described in any of the preceding embodiments, or a multispecific antibody or an antigen-binding fragment thereof as described in any of the preceding embodiments.

[0081] Taking into account the degeneracy of codons, the gene sequence encoding the above-mentioned antibody can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody; the gene can also be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0082] The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, and may refer to any recombinant polynucleotide construct that can directly introduce the target DNA fragment into the host cell by transformation, transfection or transduction to express the target gene.

[0083] On the other hand, an embodiment of the present invention provides a host cell comprising the recombinant vector described in any of the above embodiments.

[0084] Specifically, host cells include 293 cells, 293T cells, 293FT cells, CHO cells, and Per6 cells. 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins, and are well known to those skilled in the art.

[0085] On the other hand, an embodiment of the present invention further provides a method for preparing an antibody, comprising: culturing the host cell as described above to obtain the antibody. Specifically, the present invention does not specifically limit the culturing conditions of the host cell, and the culturing conditions that enable the host cell to express and produce the antibody can be obtained based on conventional technical knowledge.

[0086] On the other hand, an embodiment of the present invention provides an immunoconjugate or a pharmaceutical composition, comprising: an anti-GPRC5D antibody or an antigen-binding fragment thereof as described in any of the foregoing embodiments or a multispecific antibody or an antigen-binding fragment thereof as described in any of the foregoing embodiments.

[0087] In some embodiments, the immunoconjugate further comprises a therapeutic agent.

[0088] In some embodiments, the therapeutic agent includes at least one of a chemotherapeutic drug, a radionuclide, a photosensitizer, a photothermal agent, an immune checkpoint inhibitor, a toxin, a factor, a kinase inhibitor, an antibody against an inhibitory second signal molecule, a PD-L1 inhibitor, and a PD-1 / PD-L1 monoclonal antibody.

[0089] Relevant biomarkers for immune checkpoint inhibitor treatment include PD-L1, MSI / bMSI, TMB / bTMB, TNB, EGFR mutation, ALK fusion, TP53 mutation, and KRAS mutation.

[0090] In an optional embodiment, the above-mentioned chemotherapy drug is selected from any one or more of taxanes, vinca alkaloids, anthracyclines, epipodophyllotoxins, tyrosine kinase inhibitors, flavopiridol, irinotecan and its metabolite SN-38, topotecan, teniposide, etoposide, imatinib, gefitinib, danucetinib, doxorubicin, daunorubicin, mitoxantrone, methotrexate, camptothecin and saquinavir.

[0091] In some embodiments, the photosensitizer is selected from: (1) 5-aminolevulinic acid (ALA) or its derivatives; (2) photosensitizing compounds containing a tetrapyrrole ring; (3) traditional Chinese medicine photosensitizers; or (4) a combination of ALA or its derivatives with the compounds in (2) or (3), respectively.

[0092] In some embodiments, the photothermal agent is selected from IR-780, IR-783, IR-805, IR-808, IR-825, IR-1045, IR-1048, IR-1061, and IR-26.

[0093] In some embodiments, the inhibitory second signaling molecule can be PD-1; CTLA-4; PD-1 and CTLA-4.

[0094] In some embodiments, the PD-1 / PD-L1 monoclonal antibody drug is selected from at least one of the following groups: Nivolumab, Pembrolizumab, Pidilizumab, Lambrolizumab, BMS-936559, Atezolizumab, AMP-224, AMP224, AUNP12, BGB108, MCLA134, MEDI0680, PDROOl, REGN2810, SHR1210, STIAl lOX, STIAlllO, TSR042, BMS-936558, BGB-A317, BCD-100 and JS001.

[0095] In other embodiments, the above therapeutic agent further comprises a cytotoxic agent.

[0096] In some embodiments, the pharmaceutical composition further comprises at least one of a pharmaceutically acceptable excipient, a carrier, and a diluent.

[0097] In some embodiments, the carrier is a medically acceptable carrier, including but not limited to fillers, lubricants, disintegrants, binders, glidants, and the like.

[0098] In the preferred technical scheme of the present invention, the pharmaceutically acceptable carrier includes but is not limited to polyvinyl pyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methyl cellulose and its derivatives, ethyl cellulose and its derivatives, hydroxypropyl cellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, calcium hydrogen phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talc, indigo or a combination thereof.

[0099] In addition, the embodiments of the present invention also provide the use of the anti-GPRC5D antibody or its antigen-binding fragment as described in any of the preceding embodiments, or the multispecific antibody or its antigen-binding fragment as described in any of the preceding embodiments, or the isolated nucleic acid as described in any of the preceding embodiments, the expression cassette containing the isolated nucleic acid, or the recombinant vector containing the isolated nucleic acid, or the recombinant cell as described in any of the preceding embodiments, or the immunoconjugate or pharmaceutical composition as described in any of the preceding embodiments in the preparation of a product for preventing or treating tumors.

[0100] In some embodiments, the product includes: at least one of an immune cell, a reagent, a kit, a drug, and a pharmaceutical composition;

[0101] In some embodiments, the tumor comprises: multiple myeloma.

[0102] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0103] Example 1

[0104] (1) Preparation of GPRC5D recombinant protein:

[0105] The nucleic acid sequence encoding human GPRC5D (NM_018654.1) was synthesized by Anhui General Biotechnology Co., Ltd. PCR amplified and subcloned into the pcDNA3.1 expression vector (Invitrogen). Then, the full-length sequence of GPRC5D was cloned into the pcDNA3.1 expression vector with a His tag at the C-terminus. The 293FT cells were transiently transfected and the expression vector was expressed using FreeStyle TM The cells were cultured in shake flasks in serum-free medium (Life Technologies) for 5-7 days, and the cell pellets were collected and ultrafiltered by centrifugation. The recombinant GPRC5D protein was then purified by nickel column affinity chromatography, ion exchange chromatography, and molecular sieve chromatography.

[0106] (2) Preparation of stable cell lines expressing human GPRC5D antigen:

[0107] The full-length sequence encoding human GPRC5D was constructed into the pcDNA3.1 expression vector carrying IRES-EGFP. Hela cells were cultured in DMEM containing 10% fetal bovine serum. Cells were transfected using Lipofectamine LTX (Life Technologies) transfection reagent, flow sorted 48 hours later, cultured in 96-well plates, and monoclonal stable cell lines were screened and identified, and Hela cells stably expressing GPRC5D (GPRC5D+-Hela) were preserved.

[0108] Example 2

[0109] 1. Preparation of anti-GPRC5D monoclonal antibody.

[0110] (1) Animal immunization

[0111] 5-6 week old Balb / c female mice were used as immunized animals, and the immunization dose was 100 μg / mouse. For the first immunization, 100 μl of Freund's complete adjuvant (Sigma) was mixed with an equal volume of recombinant GPRC5D protein, and multiple subcutaneous injections were performed after sufficient emulsification. Every 2 weeks, an equal volume of Freund's incomplete adjuvant (Sigma) was mixed with the recombinant protein, and multiple subcutaneous injections were performed after sufficient emulsification. The booster immunization was performed 4 times in total. On the 10th day after the last booster immunization, the mouse antibody titer was detected by cutting the tail and collecting blood. 3 days before cell fusion, 100 μg of recombinant protein was injected into the abdominal cavity once.

[0112] (2) Cell fusion and hybridoma screening

[0113] Under sterile conditions, the spleen of mice was taken to prepare a suspension rich in B cells, and the cells were fused with SP2 / 0 according to the classic PEG (Sigma) method. The fused cells were resuspended in HAT medium for culture. Fresh HAT medium was used for half-medium replacement culture on the 5th and 10th days after fusion. ELISA, immunofluorescence and flow cytometry (FACS) analysis were performed on the 11th to 15th day after fusion to screen positive clones.

[0114] ELISA screening was performed using a 96-well plate. Briefly, GPRC5D recombinant protein was coated onto the bottom of the plate at 100 ng / well overnight at 4°C, and HRP-conjugated anti-mouse IgG antibody and chemiluminescent reagent (Beyotime Biotech) were used for color development, and the values ​​were read on a microplate reader at a wavelength of 450 nm.

[0115] Immunofluorescence staining used Hela cell lines stably expressing GPRC5D. Briefly, the cell lines were cultured in 96-well plates, 50 μl of hybridoma supernatant was added as the primary antibody, incubated at 4°C for 2 h, washed 3 times with PBS, CY3-labeled Goat Anti-Mouse IgG (Biyuntian Biotech) was used as the secondary antibody, incubated at room temperature for 1 h, washed 3 times with PBS, and images were collected using a fluorescence microscope.

[0116] Flow cytometry analysis was performed using Hela cells stably expressing GPRC5D, MM.1S cells endogenously expressing GPRC5D, and wild-type Hela cells. Briefly, cells were collected by centrifugation and resuspended in PBS buffer containing 50 μl of hybridoma supernatant, incubated at 4°C for 2 h, washed three times with PBS, and 100 μl of APC-labeled Goat Anti-Mouse IgG (BioLegend, Cat#405308) was added to each well as a secondary antibody. The cells were incubated at room temperature for 1 h, washed three times with PBS, and analyzed on a flow cytometer (Agilent-NovosamplerPro).

[0117] (3) Positive hybridoma cell subcloning

[0118] Based on the results of the above ELISA, immunofluorescence and flow cytometry experiments, the candidate clones were subcloned in a conventional manner, and then tested using the above screening method, the target positive wells were selected, and the cells were frozen.

[0119] Example 3

[0120] (4) 5 × 10E5 hybridoma cells obtained by the final screening were taken and lysed using Trizol (Invitrogen), and the total RNA of the hybridoma cells was extracted according to the standard method;

[0121] (5) Using reverse transcription (PrimeScriptTM Reverse Transcriptase, Takara), the heavy and light chain variable regions of the antibody were amplified by PCR using hybridoma sequencing primers, and then TA cloning was performed. The fragments obtained by PCR were subcloned into the PMD-19T vector. After blue-white screening, positive clones were selected for sequencing on each chain.

[0122] The following antibodies were obtained through screening.

[0123]

[0124] Example 4

[0125] Binding analysis of recombinant chimeric antibodies against HeLa cervical cancer cells overexpressing GPRC5D.

[0126] The ScFv of 16 (1#) (SEQ ID NO: 8) and the ScFv of 48 (2#) (SEQ ID NO: 16) were cloned into the pcDNA3.1 (Invitrogen) plasmid vector with the human (IgG1) heavy chain constant region (UniProtKB / Swiss-Prot: P01861.2) and transiently transfected into 293FT cells using FreeStyle TM The cells were cultured in a serum-free medium (Life Technologies) in a shake flask for 5-7 days, and the supernatant was collected and ultrafiltered by centrifugation. The corresponding anti-GPRC5D recombinant monoclonal antibodies (16(#1), 48(#2)) were then purified by Protein A / G affinity chromatography and molecular sieve chromatography columns.

[0127] GPRC5D+-Hela cells were plated in a 24-well cell culture dish. The next day, the recombinant chimeric antibody was used as the primary antibody, and CY3-labeled Goat Anti-Mouse IgG (Biyuntian Biotechnology Co., Ltd.) was used as the secondary antibody. The cells were observed and photographed using a fluorescent confocal microscope. Figure 1 As shown by Figure 1 The results showed that the recombinant chimeric antibody could specifically bind to GPRC5D+-Hela cells.

[0128] Example 5

[0129] Binding analysis of monoclonal antibodies to endogenous GPRC5D in cells.

[0130] 1. IFA detection of the binding of monoclonal antibodies 16 (#1) and 48 (#2) prepared in Example 4 to GPRC5D+-Hela cells

[0131] The stable cell line GPRC5D+-Hela cells carrying the human GPRC5D antigen constructed in Example 1 were selected and immunofluorescence analysis was performed according to the immunofluorescence method described in Example 2. The results are as follows: Figure 1 As shown, they can all bind to the GPRC5D protein on the surface of GPRC5D+-Hela cells.

[0132] 2. FACS detection of binding of monoclonal antibodies to multiple myeloma cells

[0133] GPRC5D-positive multiple myeloma cells RPMI-8226 and MM.1S cells and human acute myeloid leukemia KG-1 cells that do not express GPRC5D protein were selected and flow cytometry analysis was performed according to the flow cytometry analysis method described in Example 2. The results are as follows: Figure 2 As shown, antibodies 16 (1#) and 48 (2#) can bind well to the above cells.

[0134] Example 6

[0135] In vitro binding affinity and kinetics experiments.

[0136] This example uses the surface plasmon resonance (SPR) method for determination, and uses the GE Biacore 8K instrument for analysis. Using the kit provided by Biacore, the GPRC5D-His recombinant protein was covalently linked to the CM5 (GE) chip using the standard amino coupling method, and then the monoclonal antibodies 16 (#1) and 48 (#2) to be tested constructed in Example 4 were diluted in the same buffer according to different concentration gradients and injected. After injection, they were regenerated with the regeneration reagent in the kit. The data were analyzed and collected using the Biacore 8K supporting analysis software. The results are shown in Table 1.

[0137] Table 1. Antibody-antigen in vitro binding affinity and kinetic analysis

[0138] Sample ID KD(M) ka(1 / Ms) kdis(1 / s) Full R^2 16(1#) 8.596E-09 7.532E04 6.475E-04 0.9993 48(2#) 2.347E-10 9.747E04 2.288E-05 0.9996

[0139] Example 7

[0140] Analysis of the in vitro cytotoxic activity of bispecific antibodies against GPRC5D+-Hela cells.

[0141] The 16(1#) and 48(2#) monoclonal antibodies obtained in the above Example 4 were respectively concatenated with the anti-CD3 antibody OKT3 using (G4S)3 linker to a eukaryotic expression vector, transfected into HEK293T cells for expression, and purified using a Ni column to obtain a bispecific antibody.

[0142] GPRC5D+-Hela cells were added to a 96-well plate for non-labeled killing detection instrument (RTCA) and a common 96-well cell culture plate at 5000 cells per well and 100 μL / well (duplicate wells). The RTCA-specific 96-well plate was placed in the instrument and cultured until the Cell Index value was between 1.0 and 2.0. Then, the prepared bispecific antibody with a concentration of 0.01 nM was co-incubated with activated T cells from the peripheral blood of healthy donors at an effector-target ratio of 2:1. The RTCA monitoring time was 65 hours, and its killing activity was evaluated. The results are as follows: Figure 3 As shown, both bispecific antibodies have strong killing activity against GPRC5D+-Hela cells.

[0143] Example 8

[0144] In vitro killing experiment of anti-GPRC5D×BCMA×CD3 trispecific antibody (TsAb).

[0145] 1. Design of anti-GPRC5D×BCMA×CD3 trispecific antibody

[0146] Referring to the knob-into-hole technology, human IgG4-Fc was selected as the backbone for constructing the trispecific antibody and subjected to mutation modification, as follows:

[0147] The mutations corresponding to the knob chain in the mutation modification are: S228P, F234A, L235A and T366W, and the corresponding amino acid sequence is shown in SEQ ID No. 21;

[0148] The mutations corresponding to the hole chain in the modification are: S228P, F234A, L235A, T366S, L368A and Y407V, and the corresponding amino acid sequence is shown in SEQ ID No.22.

[0149] Based on the above design, the anti-GPRC5D single-chain antibody, BCMA nanobody and anti-CD3 antibody OKT3 were fused to the above framework to construct trispecific antibodies and named K1H1 and K2H2, respectively, where:

[0150] h1 is 48-hole, and the corresponding amino acid sequence is shown in SEQ ID No. 23; k1 is hu381-OKT3-knob, and the corresponding amino acid sequence is shown in SEQ ID No. 24;

[0151] h2 is 48-hole-hu381, and the corresponding amino acid sequence is shown in SEQ ID No. 25. k2 is 48-OKT3-knob, and the corresponding amino acid sequence is shown in SEQ ID No. 26;

[0152] The structural model of the above two heterodimeric trispecific antibodies is shown in the figure Figure 4 shown.

[0153] 2. Preparation and purification of trispecific antibodies K1H1 and K2H2

[0154] After k1, h1 and k2, h2 were mixed with transfection reagent PEI (mass-volume ratio 3:1) according to a certain mass ratio, left to stand at room temperature for 20 minutes, and slowly added to HEK293T cells in the logarithmic growth phase. After transfection for 6-8 hours, the medium was replaced with Freestyle medium and cultured for 5 days. The supernatant was collected and subjected to protein A affinity chromatography, ion exchange chromatography and molecular sieve chromatography. The results are shown in Figure 5 As shown in a and b, a is a real-time monitoring diagram of the purification process, SDS-PAGE ( Figure 5 Middle b) shows that highly pure trispecific antibodies K1H1 and K2H2 were obtained after purification.

[0155] Example 9

[0156] Binding analysis of trispecific antibodies to human GPRC5D.

[0157] The binding of the trispecific antibodies prepared in the above examples to the stably transfected cell lines GPRC5D+-Hela, BCMA+-Hela and multiple myeloma cells MM.1R was detected by FACS. Flow cytometry analysis was performed according to the method described in Example 2. The results are as follows: Figure 6 As shown, the prepared trispecific antibodies K1H1 and K2H2 have good reactivity with MM.1R, GPRC5D+-Hela and BCMA+-Hela cells.

[0158] Example 10

[0159] In vitro killing activity analysis of trispecific antibodies.

[0160] According to the method described in Example 6, the in vitro killing activity of the trispecific antibodies K1H1 and K2H2 against GPRC5D+-Hela, BCMA+-Hela and wild-type Hela cells was analyzed. The results are as follows Figure 7 As shown, the prepared trispecific antibodies K1H1 and K2H2 have strong killing activity against GPRC5D+-Hela and BCMA+-Hela cells.

[0161] Embodiment 11

[0162] In vivo testing of the antitumor activity of the trispecific antibodies K1H1 and K2H2 in a MM mouse transplant model.

[0163] MM.1S-luciferase and RPMI-8226-luciferase cells were inoculated into 6- to 10-week-old NCG mice through the tail vein at 2×106 cells per mouse. Ten days after inoculation, tumor growth was detected in a live imaging instrument and the mice were randomly divided into three groups, with 5 mice in each group. A control group was set up with an equal volume of PBS as the control group. The bispecific antibody was administered intraperitoneally at 30 μg per mouse. At the same time as the first administration, 2×106 cells were inoculated into each tail vein. 6 The bispecific antibody was administered once every 7 days for a total of 3 times. The survival status of each mouse in the experimental group (k1h1, k2h2), control group (48-OKT3, hu381-OKT3) and blank group (PBS) was observed and statistically analyzed for 60 days. The Kaplan-Meier method was used to draw the mouse survival curve and the log-rank (Mantel-Cox) test was used to statistically compare the differences in the survival of mice in each group.

[0164] The results are as follows Figure 8 As shown, the survival period of the experimental group was significantly longer than that of the control group and the PBS group, indicating that the trispecific antibody has a persistent anti-tumor activity against multiple myeloma cells.

[0165] The sequence information involved in the above examples is shown in the following table.

[0166]

[0167]

[0168]

[0169]

[0170] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An anti-GPRC5D antibody or an antigen-binding fragment thereof, characterized in that: It includes a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises: CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO: 1, 2 and 3, respectively, and the light chain variable region comprises: CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO: 5, SAS and SEQ ID NO: 6, respectively; or, The heavy chain variable region includes: CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO: 9, 10, and 11, respectively, and the light chain variable region includes CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO: 13, GTS, and SEQ ID NO: 14, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain variable region and the light chain variable region also include a framework region.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

4.

4. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

12.

5. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

7.

6. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

15.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that: The antibody is selected from any one of: a monovalent antibody, a multivalent antibody, a multispecific antibody, a chimeric antibody and a fusion antibody.

8. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The multispecific antibody includes any one of a bispecific antibody, a trispecific antibody and a tetraspecific antibody.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that: The antigen binding fragment includes any one of Fab, Fab', F(ab')2, scFv and Fv.

10. The antibody or antigen-binding fragment thereof according to claim 9, characterized in that: When the antigen-binding fragment is scFv, the amino acid sequence of scFv is shown in SEQ ID NO: 8 or 16.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that: The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region.

12. A multispecific antibody or antigen-binding fragment thereof, characterized in that: It includes: a first antigenic domain that binds to a first target; The multispecific antibody or antigen-binding fragment thereof further comprises: a second antigen domain that binds to a second target and / or a third antigen domain that binds to a third target; The first target includes GPRC5D, and the first antigen domain includes a heavy chain variable region a and a light chain variable region a; the amino acid sequence of the heavy chain variable region a is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region a is shown in SEQ ID NO:

7.

13. The multispecific antibody or antigen-binding fragment thereof according to claim 12, characterized in that: The second target includes BCMA, and the second antigen domain includes a heavy chain variable region b that binds to BCMA.

14. The multispecific antibody or antigen-binding fragment thereof according to claim 13, characterized in that: The amino acid sequence of the heavy chain variable region b is shown in SEQ ID NO:

17.

15. The multispecific antibody or antigen-binding fragment thereof according to claim 12, characterized in that: The third target includes CD3.

16. The multispecific antibody or antigen-binding fragment thereof according to claim 15, characterized in that: The third antigenic domain includes the heavy chain variable region c.

17. The multispecific antibody or antigen-binding fragment thereof according to claim 16, characterized in that: The third antigen domain also includes a light chain variable region c.

18. The multispecific antibody or antigen-binding fragment thereof according to claim 15, characterized in that: The third antigenic domain comprises a single chain antibody that binds CD3.

19. The multispecific antibody or antigen-binding fragment thereof according to claim 18, characterized in that: The amino acid sequence of the CD3-binding single-chain antibody is shown in SEQ ID NO:

18.

20. The multispecific antibody or antigen-binding fragment thereof according to claim 12, characterized in that: The number of the first antigenic domain, the second antigenic domain and the third antigenic domain is independently 1 or more than 2.

21. The multispecific antibody or antigen-binding fragment thereof according to claim 12, characterized in that: The multispecific antibody or antigen-binding fragment thereof further comprises: a heavy chain constant region and / or a light chain constant region.

22. The multispecific antibody or antigen-binding fragment thereof according to claim 21, characterized in that: The heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

23. The multispecific antibody or antigen-binding fragment thereof according to claim 21, characterized in that: The species origin of the heavy chain constant region is cow, horse, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

24. The multispecific antibody or antigen-binding fragment thereof according to claim 21, characterized in that: The heavy chain constant region includes: a knob chain and / or a hole chain.

25. The multispecific antibody or antigen-binding fragment thereof according to claim 24, characterized in that: The amino acid sequence of the knob chain is shown in SEQ ID NO:

21.

26. The multispecific antibody or antigen-binding fragment thereof according to claim 24, characterized in that: The amino acid sequence of the hole chain is shown in SEQ ID NO:

22.

27. The multispecific antibody or antigen-binding fragment thereof according to claim 12, wherein: The multispecific antibody is selected from the group consisting of: a bispecific antibody and a trispecific antibody.

28. The multispecific antibody or antigen-binding fragment thereof according to claim 12, characterized in that: The multispecific antibody comprises: an A chain and a B chain; wherein the A chain comprises the first antigen domain and a heavy chain constant region, and the B chain comprises the second antigen domain, a third antigen domain and a heavy chain constant region; the heavy chain constant region of the A chain can form a dimer with the heavy chain constant region of the B chain.

29. The multispecific antibody or antigen-binding fragment thereof according to claim 28, characterized in that: The amino acid sequence of the a chain is shown in SEQ ID NO: 23, and the amino acid sequence of the b chain is shown in SEQ ID NO:

24.

30. The multispecific antibody or antigen-binding fragment thereof according to claim 12, wherein: The multispecific antibody comprises: a C chain and a D chain; wherein the C chain comprises the first antigen domain, the second antigen domain and a heavy chain constant region; the D chain comprises the first antigen domain, the third antigen domain and a heavy chain constant region; the heavy chain constant region of the C chain can form a dimer with the heavy chain constant region of the D chain.

31. The multispecific antibody or antigen-binding fragment thereof according to claim 30, characterized in that: The amino acid sequence of the c chain is shown in SEQ ID NO:25, and the amino acid sequence of the d chain is shown in SEQ ID NO:

26.

32. An isolated nucleic acid, an expression cassette containing the isolated nucleic acid, or a recombinant vector containing the isolated nucleic acid, characterized in that: The isolated nucleic acid encodes the anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or the multispecific antibody or antigen-binding fragment thereof according to any one of claims 12 to 31.

33. A host cell, characterized in that It contains the recombinant vector described in claim 32.

34. An immunoconjugate or pharmaceutical composition, characterized in that It includes: The anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or the multispecific antibody or antigen-binding fragment thereof according to any one of claims 12 to 31.

35. The immunoconjugate or pharmaceutical composition according to claim 34, characterized in that The immunoconjugate also includes a therapeutic agent.

36. The immunoconjugate or pharmaceutical composition according to claim 35, characterized in that The therapeutic agent includes at least one of a chemotherapeutic drug, a radionuclide, a photosensitizer, a photothermal agent, an immune checkpoint inhibitor, a toxin, a kinase inhibitor, and an antibody against an inhibitory second signal molecule.

37. The immunoconjugate or pharmaceutical composition according to claim 35, characterized in that The therapeutic agent is a PD-L1 inhibitor.

38. The immunoconjugate or pharmaceutical composition according to claim 35, characterized in that The therapeutic agent is at least one of PD-1 / PD-L1 monoclonal antibody drugs.

39. The immunoconjugate or pharmaceutical composition according to claim 34, characterized in that The pharmaceutical composition further comprises at least one of a pharmaceutically acceptable excipient and a carrier.

40. The immunoconjugate or pharmaceutical composition according to claim 34, characterized in that The pharmaceutical composition also includes a diluent.

41. Use of the anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 12 to 31, the isolated nucleic acid, the expression cassette containing the isolated nucleic acid, or the recombinant vector containing the isolated nucleic acid according to claim 32, the host cell according to claim 33, or the immunoconjugate or pharmaceutical composition according to any one of claims 34 to 40 in the preparation of a product for treating multiple myeloma.

42. The use according to claim 41, characterized in that The product includes at least one of: immune cells, reagents, kits, drugs and pharmaceutical compositions.

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